The present invention relates to a method for producing cloned cows, more specifically, a method for producing somatic cell-derived cloned cows by employing in vitro maturation and subsequent enucleation of oocytes, nuclear transfer, electrofusion and activation of embryos, postactivation and in vitro culture of the embryos and transfer of the embryos into surrogate cows. It also relates to the embryos and cloned cows produced by the method described above.
Animals have long been considered to be produced by fertilization involving male and female gametes. However, tremendous efforts have been made on generating cloned animals with the identical appearance and genetic characteristics.
Cloning of zygotes has been known to be possible only in amphibians for 30 years until the success in producing a cloned offspring by substituting a pronucleus of one-cell zygote in mice(see: McGrath and Solter, Science, 220:1300-1302, 1983). Despite this first success in cloning animals, the same success in industrial animals(see: Wakayama et al., Nature, 394:369-374, 1998) has been reported much later since production of cloned mice employing mature oocytes and zygote blastomeres after 2-cell stage have several problems such as decrease in reprogramming.
With regard to the production of cloned industrial animals by nuclear transfer, it was the first report that an offspring was produced in sheep by employing blastomeres of 8- to 16-cell zygote as donor cells(see: Wiladsen, Nature, 320:63-65, 1986). Since then, only the blastomeres of zygote with totipotency by which a cell can be differentiated into every single cell have been considered to be cloned by nuclear transfer. However, through the continuous research effort, the first cloned sheep was produced by introducing nuclei from somatic cells(see: Wilmut et al., Nature, 385:810-813, 1997), thus making correction in the prior developmental theory and enabling many successful examples to be reported in production of cloned cows(see: Wells et al, Reprod. Fertil. and Develop., 10:369-378, 1998) and pigs.
The prior art methods are, however, proven to be less satisfactory in the sense that the yield of embryos produced by nuclear transfer is relatively low, which resulted in the low production rate of cloned animals.
Under the circumstances, there are strong reasons for exploring and developing an improved method for producing the somatic cell-derived embryos in terms of the yield of embryos produced by nuclear transfer.
In accordance with the present invention, it has been discovered that: the yield of embryos produced by nuclear transfer can be dramatically increased by shortening time of oocyte manipulation and improving the nuclear transfer and activation rate when donor cells are transferred into enucleated oocytes, by which somatic cell-derived cloned cows can be produced in an efficient manner.
A primary object of the present invention is, therefore, to provide a method for producing cloned cows by nuclear transfer of somatic cells.
The other object of the invention is to provide cloned cow embryos produced by the said method.
Another object is to provide somatic cell-derived cloned cows produced by the said method.